Cyclic nucleotides are known to mediate a wide variety of cellular responses to biological stimuli. The cyclic nucleotide phosphodiesterases (PDEs) are proteins which catalyze hydrolysis of 3′, 5′-cyclic nucleotides, such as cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP), to their corresponding 5′-nucleotide monophosphates. These enzymes are therefore important in control of cellular concentration of cyclic nucleotides and have a central role in a variety of intracellular signaling events, including signaling by such mechanisms and extracellular hormones, neurotransmitters and the like.
At least five distinct families of PDEs have been identified (for a review, see Beavo, Adv. in Second Mess. and Prot. Phosph. Res. 1988, 22:1–38; see also, Beavo, “Multiple Phosphodiesterase Isozymes: Background, Nomenclature and Implications”, pp. 3–15 in Cyclic Nucleotide Phosphodiesterases: Structure, Regulation and Drug Action 1990, Beavo & Houslay, Eds., John Wiley & Sons, New York; Wang et al., “Calmodulin-Stimulated Cyclic Nucleotide Phosphodiesterases”, pp. 19–59 in Cyclic Nucleotide Phosphodiesterases: Structure, Regulation and Drug Action, supra; and Manganiello et al., “Cyclic GMP-Stimulated Cyclic Nucleotide Phosphodiesterases”, pp. 62–85 in Cyclic Nucleotide Phosphodiesterases: Structure, Regulation and Drug Action, supra). These five families include: Type I or calmodulin (CaM)-stimultated PDEs; Type II or cGMP-stimulated PDEs; Type III or cGMP-inhibited PDEs; Type IV or cAMP-specific PDEs; and Type V or cGMP-specific PDEs. In addition, within each of the above-identified families there are multiple forms and isoforms (i.e., splice-variants) of closely related PDEs.
The CaM-stimulated PDEs are defined by their responsiveness to intracellular calcium levels, which leads to decreased intracellular concentrations of cAMP and/or cGMP. Indeed, increased levels of PDE activity may be observed in response to CaM in nearly every type of mammalian tissue, as well as in Drosophila, Dictyostelium and trypanosomes. Most cells therefore appear to contain at least a small amount of CaM-stimulated PDE activity. Highest levels of CaM-stimulated PDE activity have been observed in the brain and, in particular, in synaptic areas (see Greenberg et al., Neuropharmacol. 1978, 17:737–745; and Kincaid et al., Proc. Natl. Acad. Sci. U.S.A. 1987, 84:1118–1122). Several members of the CaM-PDE family have been described and are known in the art. See, for example, U.S. Pat. No. 6,015,677, issued Jan. 18, 2000 to Beavo et al.; see also, LaPorte et al., Biochemistry 1979, 18:2820–2825; Hansen et al., Proc. Natl. Acad. Sci. U.S.A. 1982, 79:2788–2792; Sharmaetal., J. Biol. Chem. 1986, 261:14160–14166; Hansen et al., J. Biol. Chem. 1986, 261:14636–14645; Snyder et al., Cell Signal 1999, 11:535–544. See also Kakkar et al., Cell Mol. Life Sci. 1999, 55:1164–1186; and Weishaar et al., J. Med. Chem. 1985, 28:537–545 for reviews.
Guanine nucleotide binding proteins (G-proteins) are also of particular interest to the background of the present invention. G-proteins are well known in the art (see, e.g., reviews by Birnbaumer, Ann. Rev. Pharmacol Toxicol. 1990, 30:675–705; and Simon et al., Science 1991, 252:802–808). Briefly, G proteins are heterotrimeric proteins, each having an α-, β- and γ-subunit, which also mediate signal transduction in a variety of different systems, including olfactory, visual, hormonal and neurotransmitter systems. G-proteins couple cell surface receptors to cellular effector enzymes, and thereby transduce an extracellular signal into an intracellular second messenger. The α-subunit of a G-protein confers most of the specificity of interaction between its receptor and effectors in the signal transduction process, while β- and γ-subunits appear to be shared among different G-proteins. Although some G-proteins (for example, Gs abd Gi) are ubiquitously expressed, others (for example transducin and gustucin) are known to be involved in sensory transduction and have been found only in specialized sensory cells (see, for example Lerea et al., Science 1986, 224:77–80; see also U.S. Pat. No. 6,008,000 issued Dec. 28, 1999 to Margolskee.
Some evidence has suggested that at least a certain G-protein may effect signal transduction by activating some, unidentified phosphodiesterase. In particular, Ruiz-Avila et al. (Nature 1995, 376:80–85) have demonstrated that a transducin-derived peptide which mimics the effects of an activated G-protein stimulates cGMP PDE activity in bovine taste lingual-tissues. However, no direct interaction between G-protein and any particular PDE has been observed.
Given, however, the wide variety of signal transduction responses in which both G-proteins and phosphodiesterases are involved and the numerous disorders associated with these different responses, there exist a need for methods to identify specific compounds that modulate signal transduction by PDEs, G-proteins or both.